1. Field of the Invention
The present invention relates to an apparatus and method capable of inspecting a specimen held on a film by irradiating the specimen with a primary beam, such as a charged-particle beam. More particularly, where the specimen consists of biological cells, the invention relates to observation and inspection of reactions of the cells when a stimulus is given to them.
2. Description of Related Art
In the fields of life science and pharmaceutics, it is important to observe reactions of biological cells produced by giving a stimulus (such as electricity, chemical substance, or medicine) to them. In the past, optical microscopes have been used for such observation. Frequently, important portions to be observed are very tiny regions of less than 0.1 μm that are impossible to observe with an optical microscope. For example, diseases arising from inability to exchange information-carrying substances normally among biological cells include hypertension, diabetes insipidus, arrhythmia, myopathy, diabetes, and deprementia. Exchange of substances among cells is performed by membrane protein molecules (such as receptors or ion channels) having sizes of about 10 nm and existing in cell membranes. Because it is difficult to observe such membrane protein molecules with optical microscopes, there has been a demand for a technique enabling observation using a scanning electron microscope (SEM) having high resolution.
However, a sample containing a specimen to be inspected with an inspection apparatus incorporating SEM capabilities is normally placed in a sample chamber whose inside pressure has been reduced by vacuum pumping. The sample placed in the sample chamber, which, in turn, is placed in a reduced-pressure ambient in this way, is irradiated with an electron beam (charged-particle beam). Secondary signals, such as secondary electrons or backscattered electrons, produced from the sample in response to the irradiation are detected. In such inspection of a sample using SEM, the sample is exposed to a reduced-pressure ambient. Therefore, moisture evaporates from the sample, so that the cells die. It has then been impossible to observe reactions to a stimulus.
Accordingly, when an inspection is performed under the condition where the sample contains moisture, it is necessary to prevent the sample from being exposed to the reduced-pressure ambient; otherwise, moisture would evaporate from the sample. One conceivable method of inspecting a sample using SEM without exposing the sample to a reduced-pressure ambient in this way consists of preparing a sample holder whose opening (aperture) for transmission of charged particles has been sealed off by a film, placing the sample in the holder, and installing the holder in an SEM sample chamber that is placed in the reduced-pressure ambient.
The inside of the sample holder in which the sample is placed is not evacuated. The film that covers the opening formed in the sample holder can withstand the pressure difference between the reduced-pressure ambient inside the SEM sample chamber and the ambient (e.g., atmospheric-pressure ambient) of the inside of the sample holder that is not pumped down. Furthermore, the film permits an electron beam to pass therethrough (see JP-T-2004-515049).
When a sample is inspected, an electron beam is directed at the sample placed within the sample holder from outside the holder via the film on the holder placed in the SEM sample chamber that is in a reduced-pressure ambient. Backscattered electrons are produced from the irradiated sample. The backscattered electrons pass through the film on the sample holder and are detected by a backscattered electron detector mounted in the SEM sample chamber. Consequently, an SEM image is derived. However, with this technique, the sample is sealed in the closed space and so it has been impossible to give a stimulus to cells from outside the sample holder, for example, using a manipulator.
An example of a method of obtaining an SEM image by preparing a film withstanding the pressure difference between vacuum and atmospheric pressure, irradiating a sample with an electron beam via the film, and detecting backscattered electrons produced from the sample in this way is described also in “Atmospheric Scanning Electron Microscopy”, Green, Evan Drake Harriman, Ph.D., Stanford University, 1993 (especially, Chapter 1: Introduction).
Examples in which two films of the structure described above are placed opposite to each other with a sample interposed between the films and in which an image is acquired by a transmission electron microscope are described in JP-A-47-24961 and JP-A-6-318445. Especially, JP-A-47-24961 also states a case in which an SEM image of the sample interposed between such films is acquired.
JP-A-2007-292702 discloses a sample inspection apparatus equipped with an open-close valve for partitioning the space between a film and primary beam irradiation means within a vacuum chamber in order to permit the sample held on the film to be exchanged quickly and to prevent contamination inside the vacuum chamber.
Morphological variations induced by the aforementioned reactions of cells to which a stimulus has been given take place in very tiny areas and so it is impossible to observe the variations with an optical microscope. Hence, SEM imaging is essential. In order to observe cells by SEM while maintaining the liquid, a sample containing the cells has been sealed in a sample holder. An electron beam has been directed at the sample via a film formed on the sample holder, thus imaging the cells.
However, the sample holder is a narrow closed space. Therefore, it has been impossible to give a stimulus to cells present within a sealed sample holder using a manipulator (i.e., manipulation).
Even if a stimulus is given to cells that are not yet sealed in by some method or other, a sequence of operations that takes several minutes or more to perform needs to be carried out. The sequence of operations consists of hermetically sealing the sample holder, putting the holder into an SEM sample chamber, pumping down the sample chamber, and irradiating the holder with an electron beam. For this reason, it has been impossible to observe cells by SEM immediately after a stimulus is given to them.
In addition, it normally takes a time of 10 seconds to 100 seconds to capture an SEM image. Consequently, it has been difficult to identify a cell portion that undergoes a morphological variation in a few seconds. Therefore, where cells respond at high speeds to a stimulus, there is the problem that SEM imaging cannot be completed.